Potyvirus infection of potato plants results in a variety of symptoms depending on the viral strain. These symptoms include production loss, leaf curling, mild mottling, rapid death of the infected area and area surrounding the infection, distorted and brittle leaves, wrinkled and rough leaves and the potato tuber necrotic ringspot disease. Necrotic ringspots render potatoes unmarketable and can therefore result in a significant loss of income. Potyviruses are transmissible by aphid vectors but may also remain dormant in seed potatoes. This means that using the same line of potato for production of seed potatoes for several consecutive generations will lead to a progressive increase in viral load and subsequent loss of crop.
Recessive resistance against specific potyvirus strains can be associated with one or several amino acid substitutions of the eIF4E protein in tomato, pepper, melon, barley, lettuce, and pea (FIG. 1). Although these substitutions appear clustered, it has proven difficult to design new versions of eIF4E mediating disease resistance.
eIF4E gene-mediated resistance is recessive, but it has been shown that the pepper gene pvr1 provides dominant potyvirus resistance when overexpressed in tomato (Kang et al., Plant Biotechnol J 5: 526-536).
It is impossible to develop potyvirus resistance in tetraploid potato by using TILLING (Piron et al., PLoS ONE 5, 2010). This method requires the use of inbred lines (which cannot be produced in tetraploid potato) and backcrossing (to segregate the trait with undesirable mutations induced by whole-genome mutagenesis treatments). Because potato is highly heterozygote and suffers from inbreeding depression, backcrossing results in reduced fitness and may trigger seedling death.
Nothing is known yet about the molecular basis of potyvirus resistance in potato and its sexually-compatible “wild potato” relatives. This lack of knowledge made it impossible to incorporate resistance into existing varieties through all-native DNA transformation, a new approach to genetic engineering that is perceived as more acceptable by consumers.
The potato eIF4E protein has a 46-amino acid domain with the consensus sequence DX1X2X3X4K SX5Q X6AW GSS X7RX8 X9YT FSX10 VEX11 FWX12X13YN NIH X14P S KLX15X16GA D (SEQ ID NO: 38), whereby either at least one of the neutral amino acids (“X”) is substituted by a charged amino acid or at least one of the charged amino acids is substituted by a neutral amino acid or an amino acid having an opposite charge. See U.S. Pat. No. 7,919,677. It is also confirmed that replacement of (i) the neutral amino acid X3 by the negative amino acid glutamate (E), or (ii) the neutral amino acid X7 by the positive amino acid arginine (R) may yield potyvirus resistance. See U.S. Pat. No. 7,772,462 B2. These studies also indicated that resistance may be obtained by replacing amino acid at position 8 by an arginine.
Potato is sexually compatible with hundreds of wild potato species, which means that it is part of an unusually large and diverse gene pool. However, despite this remarkable competitive advantage, none of the currently available potato varieties displays resistance against the agronomically-important potyvirus pathogen potato virus Y (PVY).